Abnormal tyrosine phosphorylation by the gene translocation product Bcr-Abl is the primary cause of chronic myeloid leukemia (CIVIL). One of the most successful drugs to date for CML, Gleevec, inhibits the kinase portion of the protein (Abl) and its 'normal' counterpart, c-Abl. Only a few Abl substrates have been characterized, thus the biological role of this ubiquitous regulatory protein is not well understood and its substrate interactions remain largely unknown. Understanding the role of this kinase in health and disease will provide better tools for targeting cancer. Compared to phosphorylation, thiophosphate modification is metabolically stable. Accumulation of normally transient (thio)phosphoproteins will allow the observation of phosphorylation events outside of steady-state conditions, facilitating time-resolved analysis of kinase activity. Thiophosphorylation and detection with mass spectrometry will be used to identify c-Abl and Bcr-Abl kinase substrates in healthy (non-CML) and CML cell lines. Specific aims: 1) Confirm and characterize thiophosphorylation of substrate molecules by c-Abl and Bcr-Abl; 2) Profile c-Abl and Bcr-Abl in healthy tissue and CML based on relative phosphorylation levels of Abl substrates using thiophosphoryl 'snapshots.'